Transgenic (TG) mice with overexpression of p20C/EBP[unreadable], a naturally occurring dominant negative C/EBP[unreadable] isoform, showed evidence of dentin dysplasia occurring Preferentially on the lingual side of the incisors and in the molar roots. This was characterized by reduced thickness and disrupted tubular structure of the dentin. Odontoblasts in this region were small and did not assume their characteristic elongated, polarized morphology. C/EBP[unreadable] null mice, in contrast, exhibit a tooth phenotype characterized by a spatial acceleration of incisor differentiation that appears to involve both lingual and labial surfaces. Based on a strikingly similar dentin phenotype between our TG model and fro/fro mutant mice with a mutation in the smpd3 gene that encodes neutral sphingomyelinase 2, we hypothesize a novel role for sphingolipid signaling acting through C/EBP[unreadable] in the regulation of odontoblast differentiation. The following aims are proposed: 1) To assess the temporal and spatial effects of C/EBP[unreadable] loss-of-function on tooth development using C/EBP[unreadable] TG and knockout mice. Tooth development will be assessed in these models by histology, electron microscopy, and immunohistochemical localization of endogenous C/EBP and its dimerization partner Nrf1. 2) To assess cell autonomy of the odontoblast phenotype using ex vivo dental pulp cultures and heterologous pulp transplantation. Dental pulp cultures established from wild type (WT), TG and null mice will be examined for the tempo and extent of odontoblast differentiation by assessing expression of Col1a1-promoter-driven GFP, dental sialophosphoprotein and dentin matrix protein-1 mRNA, along with the degree of mineralization of the culture. Results will be corroborated by assessing odontoblast differentiation and dentin production in dental pulp transplanted under the kidney capsule of nude mice. 3) To determine the role of the sphingomyelin signal transduction pathway on odontogenesis and the requirement for C/EBP[unreadable] function. The role of the sphingomyelin signaling pathway on odontogenesis will be assessed by testing the effects of pharmacological activators and inhibitors of the pathway on the rate and extent of odontoblast differentiation in dental pulp cultures derived from WT and mutant mice. Public Health Relevance: Understanding the transcriptional regulation of dentinogenesis will provide an essential underpinning for future tissue engineering and gene therapy approaches to reparative and regenerative dentistry using dental pulp stem/progenitor cells. The current proposal is focused on the role of the leucine zipper transcription factor C/EBP[unreadable] and the sphingomyelin signaling pathway in odontoblast differentiation. These novel, possibly interconnected pathways may have translational implications for tooth reregeneration, elucidating the mechanisms of odontoblast lineage commitment and differentiation, and the specification of highly specialized crown or root odontoblast phenotypes.